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Abstract

Human Norovirus (hNoV) is a single-stranded positive-sense RNA virus of Caliciviridae family. It is the major cause of gastroenteritis outbreaks in the United States. The VPg protein of hNoV is a multi-functional protein essential for virus replication. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of hNoV VPg regulate its nucleotidylylation. I provide evidence that hNoV VPg directly binds nucleoside triphosphates (NTPs), that inhibition of binding inhibits nucleotidylylation, and that the NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region reduces nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation through several potential mechanisms. Additionally, I demonstrate that the un-cleaved viral precursor protein, ProPol (NS5-6) is 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific role for ProPol during the hNoV infection.